Effects of telavancin on coagulation test results.

BACKGROUND: The lipoglycopeptide antibiotic, telavancin, may interfere with some laboratory coagulation tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT). OBJECTIVE: To evaluate the effects of telavancin on PT and aPTT assays in common use. METHODS: Pooled normal human plasma was spiked with telavancin 10, 20, 100 or 200 µg/ml (equivalent to trough, 2 × trough, peak and 2 × peak clinical plasma concentrations, respectively) or diluent control (0.9% sodium chloride). Samples were analysed using 16 PT reagents and seven aPTT reagents. RESULTS: Telavancin 200 µg/ml (corresponding to 2 × peak clinical plasma concentration), produced significant PT prolongation (> 9% difference vs. diluent control) with all the 16 PT reagents (range 12% to > 600%). At lower telavancin concentrations, PT prolongation was dose-dependent and varied among reagents, but appeared greatest with preparations containing recombinant tissue factor. With telavancin 10 µg/ml (equivalent to trough), PT prolongation was 10% with HemosIL(®) PT-Fibrinogen Recombinant, while ranging from 5% to -1% with all other reagents. Significant (> 34% difference vs. baseline) and dose-dependent aPTT prolongation was observed with all the seven reagents in samples spiked with telavancin 100 or 200 µg/ml (range 65-142% at 200 µg/ml). aPTT reagents containing a silica activator appeared to be more sensitive to telavancin interference. Telavancin 10 µg/ml was not associated with increased aPTT with any of the reagents tested. CONCLUSIONS: Telavancin has the potential to prolong both PT and aPTT in vitro. It is recommended that samples for PT or aPTT be obtained just prior to a telavancin dose (trough).

Structure-activity relationship studies of a bisbenzimidazole-based, Zn(2+)-dependent inhibitor of HCV NS3 serine protease.

A survey of isosteric replacements of the phosphonoalanine side chain coupled with a process of conformational constraint of a bisbenzimidazole-based, Zn(2+)-dependent inhibitor of hepatitis C virus (HCV) NS3 serine protease resulted in the identification of novel series of active compounds with extended side chains. However, Zn(2+)-dependent HCV NS3 inhibition was relatively insensitive to the structural variations examined but dependent on the presence of negatively charged functionality. This result was interpreted in the context of an initial electrostatic interaction between protease and inhibitor that is subsequently consolidated by Zn(2+), with binding facilitated by the featureless active site and proximal regions of the HCV NS3 protein.

Peptidyl diazomethyl ketones inhibit the human rhinovirus 3C protease: effect on virus yield by partial block of P3 polyprotein processing.

The efficacy of a series of diazomethyl ketones (DMKs) was measured in rhinovirus-infected cultures and against the HRV14 3C protease. Their specificity and potency were confirmed against purified recombinant enzyme expressed in a yeast secretion system. An internally quenched fluorescent peptide substrate was used to assess the potency against the enzyme, obtaining a 50% inhibitory concentration (IC50) of 1 microM for both Z-L-F-Q-CHN2 and Z-V-L-F-Q-CHN2, while a lower affinity was observed for Z-F-Q-CHN2. The tripeptide Z-L-F-Q-CHN2 blocked viral replication with an IC50 value of 30 microM as judged by the reduction in viral induced cytopathy of HeLa-H1 cells, as well as a marked reduction in viral plaque formation (50% effective concentration=20 microM). Western blot analysis of viral proteins from infected cells indicates that this inhibitor works specifically by blocking viral polyprotein maturation, displaying a reduction of detectable 3C protease and an accumulation of the 3CD polypeptide. These results indicate that DMK inhibitors of the 3C protease have antiviral potency. Furthermore, the pattern of viral protein processing observed suggests that reducing the concentration of mature HRV 3C protease even in the presence of increased 3CD protein is sufficient to block proper viral processing and significantly reduce virus yield.

Synthesis of a statistically exhaustive fluorescent peptide substrate library for profiling protease specificity.

A statistically exhaustive, 8800 compound tripeptidal amidomethylcoumarin library was synthesized as discreet compounds using solid-phase combinatorial chemistry. A subset of the compounds was purified by HPLC and tested in a high-throughput fluorometric assay against several known serine and cysteine proteases to demonstrate the utility of this library for profiling protease substrate specificity.

High-throughput screening of enzyme inhibitors: simultaneous determination of tight-binding inhibition constants and enzyme concentration.

Active site titration by a reversible tight-binding inhibitor normally depends on prior knowledge of the inhibition constant. Conversely, the determination of tight-binding inhibition constants normally requires prior knowledge of the active enzyme concentration. Often, neither of these quantities is known with sufficient accuracy. This paper describes experimental conditions under which both the enzyme active site concentration and the tight-binding inhibition constant can be determined simultaneously from a single dose-response curve. Representative experimental data are shown for the inhibition of human kallikrein.

High-throughput screening of enzyme inhibitors: automatic determination of tight-binding inhibition constants.

Determination of tight-binding inhibition constants by nonlinear least-squares regression requires sufficiently good initial estimates of the best-fit values. Normally an initial estimate of the inhibition constant must be provided by the investigator. This paper describes an automatic procedure for the estimation of tight-binding inhibition constants directly from dose-response data. Because the procedure does not require human intervention, it was incorporated into an algorithm for high-throughput screening of enzyme inhibitors. A suitable computer program is available electronically (http://www.biokin.com). Representative experimental data are shown for the inhibition of human mast-cell tryptase.

A novel approach to serine protease inhibition: kinetic characterization of inhibitors whose potencies and selectivities are dramatically enhanced by Zinc(II).

Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.

Purification and characterization of clavaminate synthase from Streptomyces antibioticus. A multifunctional enzyme of clavam biosynthesis.

Clavaminate synthase (CS), a key enzyme in the clavulanic acid biosynthetic pathway, has been purified to electrophoretic homogeneity from Streptomyces antibioticus (Tü 1718), a species that does not produce clavulanic acid. A comparison of the physical and kinetic properties of clavaminate synthase from S. antibioticus (CS3) and the two isozymes from Streptomyces clavuligerus (CS1 and CS2) has been conducted. In oxidative reactions requiring the co-substrates O2, alpha-ketoglutaric acid, and catalytic Fe2+, both CS1 and CS2 catalyze three distinct transformations, the hydroxylation of deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and the cyclization and desaturation of proclavaminic acid to clavaminic acid. We have demonstrated that CS3 from S. antibioticus also catalyzes these three oxidations. The apparent molecular mass of CS3 from matrix-assisted laser desorption mass spectrometry is 35,839 +/- 36 Da. The enzyme is a monomer in solution as determined by gel filtration chromatography. Analysis of the four possible proclavaminic acid diastereomers confirmed the absolute configuration of the substrate to be 2S,3R. Based upon N-terminal sequence comparisons among the three proteins, CS3 possesses the higher degree of homology with the CS1 isozyme from S. clavuligerus. Although previously associated solely with clavulanic acid biosynthesis, we propose these findings and recent precursor incorporation data support the view that clavaminate synthase plays a critical role in the biosynthesis of the clavam metabolites.

A kinetic investigation of phosphoenolpyruvate carboxylase from Zea mays.

The reaction catalyzed by phosphoenolpyruvate carboxylase from Zea mays has been studied kinetically. Results of initial velocity patterns and inhibition studies indicate that phosphoenolpyruvate carboxylase has a random sequential mechanism in which there is a high level of synergism in the binding of substrates. The preferred order of addition of reactants is Mg2+, phosphoenolpyruvate, and bicarbonate. The binding of Mg2+ is at equilibrium. Values for the various kinetic parameters are KiMg = 2.3 +/- 0.4 mM, KPEP = 3.6 +/- 0.6 mM, KiPEP = 0.2 +/- 0.07 mM, and Kbicarbonate = 0.18 +/- 0.04 mM. In addition, double inhibition experiments have been performed to examine the nature of the active site interactions with the putative intermediates, carboxy phosphate and the enolate of pyruvate. Highly synergistic inhibition of phosphoenolpyruvate carboxylase was observed in the presence of oxalate and carbamyl phosphate (alpha = 0.0013). However, an antisynergistic relationship exists between oxalate and phosphonoformate (alpha = 2.75).

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates.

The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (Z)- and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of 18O-labeled bicarbonate produces phosphate that is multiply labeled with 18O; in addition, 18O is also incorporated into residual (Z)- and (E)-F-PEP. The 13(V/K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 degrees C, is 1.049 +/- 0.003 for (Z)-F-PEP and 1.009 +/- 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on CO2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of CO2, which results in hydrolysis of PEP.

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays utilizing formate as an alternate substrate for bicarbonate.

Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.